1. Field of the Invention
This invention relates generally to the formulation of microorganisms for delivery of viable inoculum of the microorganisms to an environment. This invention also relates generally to the formulation of microorganisms for delivery of viable inoculum of the microorganisms to an environment having an indigenous microflora and to a polluted environment or site. Methods of formulating viable inoculum of microorganisms for delivery of the microorganisms to a polluted environment or site, and a method for remediation of a polluted environment or site are presented, in which a suitable microorganism having the capacity to decompose a pollutant, is applied to the site in combination with a suitable carrier for the microorganism.
The invention also relates to compositions for remediating an environment or site which has been contaminated with a chemical pollutant, and to methods for delivering microorganisms in combination with a carrier for the microorganisms to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation. Methods are presented for delivering nutrients in combination with a carrier for the nutrients to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation, as are methods for delivering nutrients in combination with a microorganism and a carrier to an environment or site which has been contaminated with a chemical pollutant and which is subject to bioremediation.
Embodiments of the invention are presented relating to the biodegradation of benzo[a]pyrene, and to the fungus Marasmiellus troyanus. In particular, Marasmiellus troyanus isolate no. 216-1867 is presented, and compositions comprising M. troyanus isolate no. 216-1867 in combination with a carrier are presented. Yet further, the invention relates to the degradation of benzo[a]pyrene by M. troyanus, and the mineralization of benzo[a]pyrene by M. troyanus. A process for bioremediation of polluted media contaminated with benzo[a]pyrene is also shown.
2. Background of the Related Art
Chemical pollution of various media (e.g. soil, water) is a common problem worldwide which has a major economic impact at the local, national, and global levels. The remediation of sites polluted or contaminated with toxic chemicals or hazardous wastes using existing technologies is generally extremely costly, laborious and time-consuming. For example, in the U.S., Congress established a multibillion dollar fund (the Superfund) under the Comprehensive Environmental Response, Compensation, and Liability Act of 1980, 1986 and 1990 (commonly known as the Superfund Act). The fund was established to pay for cleanup of polluted sites such as hazardous and municipal waste dumps, contaminated factories and mines, and leaking underground fuel storage tanks. During the eleven years between its inception and 1991, 30,000 potential Superfund sites were surveyed. The time required for cleanup of a Superfund site by the EPA has been as long as 10 years. Remediation efforts at hazardous waste sites have been partially effective 54% of the time and completely effective only 16% of the time (Riser-Roberts, E., (1992) "Bioremediation of Petroleum Contaminated Sites," pp. 1-34, CRC Press, Boca Raton, Fla.). Currently, additional sites of chemical pollution are being discovered and new sites of pollution are being created worldwide.
Pollution of the soil with toxic chemicals creates hazards to the health of humans and other organisms, and also renders the site useless for most purposes. In addition, pollutants in the soil can be leached into underlying aquifers leading to groundwater contamination. In the U.S., groundwater is used as a source of drinking water by about 120 million people, and is also widely used to irrigate food crops.
Improved technologies for remediation of polluted soil and water are urgently needed. Traditional methods for cleanup of contaminated soil has generally involved excavation of the soil, followed by treatment or containment. Currently used techniques for remediating polluted soils include, for example, the physical removal of volatile materials by aspiration (vacuum extraction) and the incineration of contaminated soil. Because of the large volumes of soil usually involved, physicochemical methods such as those exemplified above may be prohibitively expensive. An alternative approach to cleaning up sites of chemical pollution is bioremediation, in which a biological organism is used as an agent for converting the chemical pollutant to less toxic or nontoxic compounds. For example, various microorganisms have been found to detoxify a number of toxic chemical pollutants (see, for example, G. Chaudry (Ed.) "Biological degradation & bioremediation of toxic chemicals," Dioscorides Press, Portland, Oreg., 1984). Biodegradation or detoxification of chemical pollutants is normally the result of one or more enzymatic reactions, including oxidation, reduction, hydrolysis, and conjugation (see, for example, D. W. Connell, & G. J. Miller, (1984) "Chemistry & Ecotoxicology of Pollution," pp. 1-48 & 231-247, John Wiley & Sons, Inc., New York, N.Y.).
One factor limiting the efficacy of prior art bioremediation processes is the tendency of microorganisms to lose viability and decline in number following their introduction to the remediation site. It has been demonstrated by numerous field trials that, in general, microorganisms released into the soil tend not to spread from the point of application, and further that their numbers tend to decline over time (see, for example, J. D. van Elsas, & C. E. Heijnen, "Methods for the introduction of bacteria into the soil: A review," Biol. Fertil. Soils, 10:127-133, 1990). Factors militating against the propagation and survival of microorganisms introduced into soils include: competition with other organisms for nutrients, water and space; parasitism, antibiosis and predation by other organisms; and unfavorable physicochemical parameters of the soil milieu, including sub-optimal pH, water and oxygen concentrations. In the case of polluted soils, problems associated with survival and propagation of microorganisms introduced into such soil may be exacerbated by the presence of toxic pollutants at concentrations which are inimical to microbial growth.
In an attempt to prolong the survivability of microorganisms introduced into soil, some prior art remediation techniques have incorporated increased aeration or the large-scale application of nutrients to the soil. However, this approach is expensive and, in addition, nutrients added to the soil en masse are immediately available to the soil microflora as a whole, and consequently are prone to rapid depletion.
Another approach to increasing survivability of microorganisms introduced into soil, or other environments, is to combine the microorganisms with various carrier materials. Such carriers include a variety of organic and inorganic materials, including silica, mineral oil, peat, and various gels (see, for example, D. A. Van Schreven, "Some factors affecting growth and survival of Rhizobium spp. in soil-peat cultures," Plant & Soil, 32:113-130, 1970; Fravel, D. R., et al., "Encapsulation of potential biocontrol agents in an alginate-clay matrix," Phytopathology, 75:774-777, 1985; W. J. Connick, Jr., "Formulation of living biological control agents with alginate," in B. Cross & H. B. Scher (eds.) "Pesticide formulations: innovations & developments," American Chemical Society, Washington, D.C., pp. 241-250, 1988).
Apart from a carrier in combination with a microorganism serving as a vehicle for the microorganism, in some cases the particular combination of a suitable carrier material with a certain microorganism provides the added advantage of preserving the microorganism, thereby allowing for storage of the microorganism in a viable state (see, for example, D. J. Daigle & P. J. Cotty, "Formulating atoxigenic Aspergillus flavus for field release," Biocontrol Science & Technology, 5:174-184, 1995; D. R. Fravel, et al., "Encapsulation of potential biocontrol agents in an alginate-clay matrix," Phytopathology, 75:774-777, 1985; W. J. Connick, Jr., "Formulation of living biological control agents with alginate," in B. Cross & H. B. Scher (eds.) "Pesticide formulations: innovations & developments," American Chemical Society, Washington, D.C., pp. 241-250, 1988).
As mentioned above, in numerous field trials microorganisms have been released into the soil but their survival has been limited and their effectiveness poor due to the complex interactions and harsh conditions within the soil environment (J. D. van Elsas, & C. E. Heijnen, "Methods for the introduction of bacteria into the soil: A review," Biol. Fertil. Soils, 10:127-133, 1990). Techniques for in situ bioremediation of polluted soil can profit from lessons learned in nitrification, biocontrol, and other areas of applied microbiology in which living microorganisms are introduced into the environment. For example, to address the problems of lack of survival and effectiveness, microorganisms have been formulated with various carriers and encapsulating agents, and the formulations applied to the particular environment with varying results. One such carrier or encapsulating agent that has been used to encapsulate various fungi is alginate, a naturally occurring .beta.-1,4-linked copolymer of .alpha.-L-glucuronate and .beta.-D-mannuronate. Alginate gel is non-toxic and biodegradable, making alginate gel beads well-suited as vehicles for the release of microorganisms, nutrients, etc., into the environment.
Livernoche et al., describe the use of alginate-encapsulated white rot fungus, Coriolus versicolor, to decolorize kraft mill effluents containing lignin (D. Livernoche, et al., "Decolorization of a Kraft mill effluent with fungal mycelium immobilized in calcium alginate gel," Biotechnol. Lett. 3:701-706, 1981). Alginate-encapsulated C. versicolor has also been used as mushroom spawn.
Various fungi have been combined with carrier materials for application to plant surfaces, soil, or soil surfaces for the purposes of biological pest control. For example, U.S. Pat. No. 4,718,935 to Walker et al. discloses the encapsulation of mycoherbicidal fungi in alginate gel pellets as a form of mass-produced inoculum for the control of weeds.
U.S. Pat. No. 4,724,147 to Marois et al. discloses the encapsulation of fungi in alginate pellets as inoculum for the control of soil-borne plant diseases in agriculture.
U.S. Pat. No. 4,668,512 to Lewis et al. discloses the formulation of fungi with wheat bran to form alginate gel pellets, for the control of soilborne plant pathogens, wherein the wheat bran provides a nutrient source for the fungus.
None of the patents cited above, alone or in combination, teaches formulation of microorganisms having the capacity to degrade chemical pollutants, or the use of such organisms for bioremediation. Furthermore, the use of wheat bran in a fungal inoculum formulation, as taught by the process of Lewis et al., is not suitable for some fungi. Thus, we have found that the white rot fungus Phanerochaete chrysosporium formulated with wheat bran did not yield growth of the fungus after the wheat bran-formulated fungus was plated on a nutrient medium. Moreover, our subsequent studies have demonstrated that wheat bran, as well as other wheat products including purified wheat gluten, actually caused inhibition of growth of P. chrysosporium growing from alginate gel beads formulated without wheat bran. On the other hand, inhibition of growth of P. chrysosporium or other fungi has not been observed when sawdust, corncob grits, Pyrax clay or any other non-wheat based materials were used to formulate fungal inoculum according to the instant invention.
Connick, Jr. discloses in U.S. Pat. Nos. 4,401,456 and 4,400,391 processes for the incorporation of biocidal chemical compounds, such as insecticides and herbicides, into alginate gels. Certain biocidal compounds, e.g. some pesticides, are known as environmental pollutants and as such are potentially subject to various bioremediation techniques, including the techniques disclosed herein. U.S. Pat. Nos. 4,401,456 and 4,400,391 do not teach the encapsulation of fungi or other organisms.
Mitchell, in U.S. Pat. No. 3,649,239 teaches formulations of fertilizers, either as solutions or emulsions, in combination with alginate for the purpose of slow release of fertilizer to soil. U.S. Pat. No. 3,649,239 does not teach formulation of microbial inoculum, nor encapsulation of fungi or of other organisms.
U.S. Pat. No. 5,085,998 to Lebron et al. discloses the degradation of 2,4,6-trinitrotoluene (TNT) by contacting either a liquid culture or a soil-corncob culture of P. chrysosporium with a quantity of TNT. Lebron et al. do not disclose encapsulation of fungi within alginate, nor the degradation of polyaromatic hydrocarbons or other pollutants other than TNT.
U.S. Pat. No. No. 5,278,058 to Call discloses the production of lignolytical (lignolytic) enzymes by Phanerochaete chrysosporium cultured in a fermentation vessel which is agitated by rotating and slewing movements of the vessel. U.S. Pat. No. 5,278,058 does not disclose the encapsulation of fungi in a carrier, vehicle or gelling agent.
Matsumura et al., U.S. Pat. No. 5,342,779 discloses the use of Phanerochaete chrysosporium for the degradation of halogenated organic compounds in a polluted medium by contacting the medium with the fungus, and simultaneously exposing the medium to ultraviolet radiation. Matsumura et al. do not teach the encapsulation of a microorganism with a carrier, vehicle or gelling agent.
Komatsu et al., EP publication No. 0646642 A2, discloses a microorganism (bacteria) in combination with a carrier and an inducer of a degradative enzyme, in which the microorganism is adsorbed on a surface of a carrier or on a surface of a water-absorbent polymer, and the microorganism and the carrier form an integral unit in that the microorganism remains in permanent association with the carrier. Thus, Komatsu et al. do not teach encapsulation of microorganisms with a carrier wherein the microorganism grows from the carrier and propagates itself within the medium in the absence of the carrier.
None of the patents or publications cited above teach the application of a microorganism formulated as an alginate bead to contaminated soil or to any other medium having toxic chemical pollutants therein, wherein the organism can remain viable within the alginate bead carrier for an extended period of time following application to the polluted medium; and the organism may grow away from the alginate bead carrier into the surrounding polluted medium and propagate itself, in the absence of the carrier, within the polluted medium; thereby the organism effectively colonizes the polluted medium into which the alginate bead formulation of the organism was introduced. Indeed, the ability to maintain a viable culture within the soil environment for a long enough duration, and in proximity to the target pollutant(s), has been the limiting factor in most bioremediation applications to date.
The degradation of toxic pollutants by many microorganisms, and particularly by bacteria, requires the pollutant to be not only in contact with or proximate to the microorganism, but also to be taken up by the cell where it can interact with intracellular degradative enzyme(s). White rot fungi, on the other hand, can perform this remedial function by the release of extracellular enzymes, such as ligninases. The mechanisms of enzymic degradation exhibited by many lignicolous microorganisms are non-specific and non-stereo selective, thereby increasing the metabolic versatility of such organisms and making them highly advantageous for bioremediation of sites contaminated with various pollutants. Likewise the considerable latitude in formulating degradative organisms as disclosed herein, increases the likelihood that any given degradative microorganism will have the ability to survive within, and colonize, a particular polluted medium subject to remediation.
Although to date, the majority of microorganisms used for bioremediation have been bacteria (prokaryotes), fungi (eukaryotes) are also potentially valuable in this regard. For example, in vitro laboratory studies with the lignolytic white rot fungi, such as Phanerochaete chrysosporium, have shown the ability of these fungi to degrade a range of toxic compounds including polychlorinated biphenyls (PCBs), chlorinated pesticides, polyaromatic hydrocarbons, such as benzo[a]pyrene, pyrene, phenanthrene, fluorene, and nitroaromatic compounds (e.g. trinitrotoluene, TNT). D. P. Barr & S. D. Aust "Mechanisms white rot fungi use to degrade pollutants," Environmental Science Technology 28:78A-87A, 1994; J. A. Bumpus et al. "Oxidation of persistent environmental pollutants by a white rot fungus," Science 220:1434-1438, 1985. The ability of Phanerochaete spp. to degrade a range of structurally unrelated compounds has been ascribed largely to their lignolytic metabolic activity (S. D. Aust "Degradation of environmental pollutants by. Phanerochaete chrysosporium," Microbial Ecology, 20:197-209, 1990).
Despite the ability of P. chrysosporium and other lignolytic fungi to degrade numerous toxic pollutants, there has been little commercial success in applying these organisms to bioremediation. A major factor limiting the use of P. chrysosporium in bioremediation is the difficulty of introducing inoculum of the fungus to a contaminated medium in a manner that enables the fungus to grow and propagate within the medium. However, P. chrysosporium is not alone in this regard. On the contrary, it is normally problematic to establish recently-introduced microorganisms in a medium having an established indigenous microflora. The compositions and methods of the instant invention are designed to overcome the difficulty of establishing an introduced microorganism in such a medium, and to allow the successful use of a number of potentially valuable degradative organisms in bioremediation.
A common chemical pollutant of soil which has been reported from industrial sites worldwide is benzo[a]pyrene (see, for example, A. C. Johnson & D. Larsen "The distribution of polycyclic aromatic hydrocarbons in the surficial sediments of Penobscot Bay (Me., USA) in relation to possible sources and to other sites worldwide," Mar. Environ. Res., 15:1-16, 1985). Benzo[a]pyrene is one of a large number of polybenzenoid hydrocarbons. The structure of benzo[a]pyrene is depicted in FIG. 1A. Benzo[a]pyrene is one of the most potent known carcinogens within the polycyclic aromatic hydrocarbons. The actual carcinogen is a diol epoxide metabolite of benzo[a]pyrene resulting from enzymatic oxidation of benzo[a]pyrene which has entered the body. The diol epoxide (FIG. 1B) alkylates DNA leading to mutations. The accumulation of benzo[a]pyrene is therefore a threat to the environment and public health.
Benzo[a]pyrene occurs in crude petroleum, in petroleum products such as coal tar, and in fossil fuel by-products such as soot. Most accumulations of benzo[a]pyrene in the environment result, directly or indirectly, from the incomplete combustion of fossil fuels. Benzo[a]pyrene is extremely persistent in the environment due to its low solubility, chemical stability and resistance to degradation by most organisms. Indeed, many hydrocarbon-degrading bacteria are unable to break apart the fused benzene rings of benzo[a]pyrene. Further, those bacteria which have the ability to degrade benzo[a]pyrene have proved difficult to establish at contaminated sites.